Emulsion and micellar formulations for the delivery of biologically active substances to cells

ABSTRACT

New emulsion and micelle formulations are described as are complexes of these formulations with biologically active substances. The novel formulations are different from cationic lipid vectors such as cationic liposomes in that the complexes formed between biologically active substances and the emulsion and micellar formulations of this invention are physically stable and their transfection activity is resistant to the presence of serum. These novel formulations are disclosed to be useful in areas such as gene therapy or vaccine delivery.

FIELD OF THE INVENTION

[0001] The present invention relates to the use of lipid dispersions todeliver biologically active substances to cells. In particular, thepresent invention relates to emulsion and micellar formulations and tothe ability of these formulations to form stable complexes withbiologically active substances and thereby facilitate the delivery ofthese substances to cells.

BACKGROUND OF THE INVENTION

[0002] Cationic liposomes are of interest as a non-viral vehicle for thedelivery of biologically active substances such as drugs, hormones,enzymes, nucleic acids and antigens, including viruses, to cells both invitro and in vivo. Indeed, cationic liposomes have been demonstrated todeliver genes in vivo (Nabel, E. G., et al. (1990) Science, 249:1285-1288), (Brigham, K. L., et al. (1989) Am. J. Respir. Cell Mol.Biol., 195-200, Stribling, R., et al. (1992) Proc. Natl. Acad. Sci.U.S.A., 89: 11277-11281), (Plautz, G. E., et al. (1993) Proc. Natl.Acad. Sci. U.S.A., 90: 4645-4649, Stewart, M. J., et al. (1992) Hum.Gene Ther., 3: 267-275). However, the inhibition by serum components ofthe transfer of nucleic acids by cationic liposomes limits theapplication of liposomes as a vector for nucleic acids in vivo toregional administrations which avoid exposure to serum.

[0003] In addition, stability is a major problem limiting the use ofliposomes, both in terms of shelf life and after administration in vivo.Thus, it is desirable to explore the use of other types of lipiddispersions as delivery systems utility for biologically activesubstances.

[0004] U.S. Pat. No. 4,610,888 refers to the use as a drug-deliverysystem of water-in-oil emulsions in which the volume of aqueous phaseranges from about 0.7% to about 10.25% of the volume of the lipidcomponents used. However, such water-in-oil emulsions are unsuitable fordelivering substances in blood or in other aqueous body tissues.

SUMMARY OF INVENTION

[0005] The present invention relates to novel emulsion and micellarformulations useful for delivering biologically active substances tocells. The emulsion and micellar formulations of this invention arecompatible with blood, retain activity in the presence of serum and arestable in storage. The emulsions of this invention comprise lipidcomponents and an aqueous carrier, where the lipid components comprisean oil component, a cationic amphiphile component, preferably a cationiclipid, and a nonionic surfactant component. The micellar formulationscomprise lipid components and an aqueous carrier, where the lipidcomponents comprise a cationic amphiphile component and a nonionicsurfactant component. The lipid components of the emulsion and micellarformulation of the present invention may further comprise a neutralphospholipid component.

[0006] “Component” as used throughout the specification and claims isdefined as: comprising at least one cationic amphiphile or a mixture ofamphiphiles when used in the phrase “amphiphile component”; comprisingat least one oil or a mixture of oils when used in the phrase “oilcomponent”; comprising at least one nonionic surfactant or a mixture ofnonionic surfactants when used in the phrase “nonionic surfactantcomponent”; comprising at least one neutral phospholipid or a mixture ofneutral phospholipids when used in the phrase “neutral phospholipidcomponent”.

[0007] The invention further relates to complexes formed by combiningbiologically active substances and the above-identified emulsion andmicellar formulations. These biologically active substance:emulsion andbiologically—active substance:micelle complexes are stable over time andmay have therapeutic and/or prophylactic utility in vivo depending onthe activity of the biologically active substance contained in thecomplex.

[0008] This invention also provides a method for delivering abiologically active substance to cells by exposing cells to thecomplexes of this invention. In one embodiment, a method of exposingcells to a biologically active substance is provided, said methodcomprising culturing said cells in the presence of a biologically activesubstance:emulsion complex or a biologically active substance:micellecomplex thereby facilitating delivery of the biologically activesubstance to cells.

[0009] The invention further provides a method of delivering abiologically active substance to cells in vivo comprising administeringto an animal or human the complexes of this invention. It is to beunderstood that the complexes used for the delivery of biologicallyactive substances to cells in vitro or in vivo may be freshly preparedby admixture or may be prepared earlier and stored prior to their use.

[0010] The invention further relates to a kit containing an emulsion ormicellar formulation of the present invention. The invention alsoprovides a kit containing a complex formed between a biologically activesubstance and an emulsion or micellar formulation of the presentinvention.

[0011] Methods for producing emulsion and micellar formulationsaccording to the invention are also'provided herein.

[0012] In one embodiment, a method for producing an emulsion formulationof this invention comprises

[0013] a) combining an organic solvent with an oil component, a cationicamphiphile component and a nonionic surfactant component;

[0014] b) removing the organic solvent to leave a lipid film; and

[0015] c) suspending the lipid film in an aqueous carrier to producesaid emulsion formulation miscible in aqueous solution.

[0016] In an alternative embodiment, the oil may serve as the organicsolvent in step (a) such that the method for producing an emulsionformulation of this invention comprises

[0017] a) combining an oil component, a cationic amphiphile componentand a nonionic surfactant component; and

[0018] b) adding an aqueous carrier to the combination of step (a).

[0019] When a neutral phospholipid component is to be included in theemulsion, the neutral phospholipid component is combined with the abovecomponents in step (a).

[0020] The method for producing a micellar formulation miscible inaqueous solution comprises:

[0021] a) combining an organic solvent with a cationic amphiphilecomponent and a nonionic surfactant component;

[0022] b) removing the organic solvent to leave a lipid film; and

[0023] c) suspending the lipid film in an aqueous carrier to producesaid micellar formulation miscible in aqueous solution.

[0024] When a neutral phospholipid component is to be included in themicellar formulation, the neutral phospholipid component is combinedwith the above components in step (a).

DESCRIPTION OF FIGURES

[0025]FIG. 1 shows the optimization of transfection of BL6 cells withcomplexes formed between pCMV-Luc DNA and formulations #21 (-), #27(▪-▪), #28 (▴-▴) or #30 (◯-◯) (see Table 3 for compositions offormulations) by mixing 6 μl of each formulation (6 μl of eachformulation contained 4.5 μg of DC-Chol) with varying amounts ofpCMV-Luc DNA as indicated on the horizontal axis.

[0026]FIG. 2 shows the optimization of transfection of BL6 cells withcomplexes formed between pCMV-Luc DNA and varying amounts offormulations #21 (-), #27 (▪-▪), #28 (▴-▴) or #30 (◯-◯) as indicatedon the horizontal axis (see Table 3 for compositions of formulations).The amount of pCMV-Luc DNA was fixed at 2 μg for formulations #27 and#30 and at 1.5 μg pCMV-Luc DNA for formulations #21 and #28 and “μgformulation” on the horizontal axis refers to μgs of total lipidcomponents present in the amount of formulation combined with pCMV-LucDNA to form complex.

[0027]FIG. 3 shows the stability of the complexes formed between DNA andthe indicated emulsion or micellar formulations (see Table 3 forcomposition of formulations). Complex was prepared with 2 μg of pCMVCATand 16 μl of the indicated formulations containing the same amount ofDC-Chol (12 μg) in a final volume of 250 μl, except for the DC-Chol/DOPEliposome:DNA complexes which were prepared with 1 μg pCMVCAT and 6 μgliposome in a final volume of 250 μl.

[0028]FIG. 4 shows the effect of varying the concentration of Tween 80in emulsions containing 0.25 mg oil, 0.25 mg DOPE, 0.75 mg DC-Chol and xmg Tween 80 per ml on the average diameter of concentrated ((-) anddiluted (◯-◯) pCMV-Luc DNA/emulsion complexes.

[0029]FIGS. 5A and 5B show the effect of Tween 80 on thetransfection/activity of concentrated (FIG. 5A) and diluted (FIG. 5B)pCMV-Luc DNA/emulsion complexes in BL6 cells in medium containing either0 or 20% serum. The emulsion formulations used to produce the dilutedand concentrated DNA:emulsion complexes contained 0.25 mg oil, 0.25 mgDOPE, 0.75 mg DC-Chol and varying amounts of Tween 80 per ml.Concentrated DNA/emulsion complex was formed by adding 2 μl of solutioncontaining 8 μg of pCMV-Luc DNA to 72 μl of emulsion and dilutedDNA/emulsion complex was formed by combining 2 μg of pCMV-Luc DNA in 125μl with 18 μl of emulsion diluted to 125 μl.

[0030]FIG. 6 shows CAT reporter gene expression in mice injected via thetail vein with DNA:emulsion or DNA:micelle complexes. The complexes wereformed as follows: 200 μl each of 4× concentrates of formulations #21(1100 μg total lipid components), #28 (1000 μg total lipid components),#34 (900 μg total lipid components) and #31 (700 μg total lipidcomponents) were mixed with 6 μl of 5M NaCl to a final concentration ofNaCl of 0.15M and then combined with 25 μl of 4 μg/μl pCMV-CAT DNA (100μg). The amount of DC-Chol contained in each DNA:emulsion andDNA:micelle complex was 600 μg. After two days, organs were excised andprotein was extracted. CAT activity was measured by using 0.1 μCi [¹⁴C]chloramphenicol as substrate. Each bar represents the mean of two mice.

DETAILED DESCRIPTION OF INVENTION

[0031] The present invention relates to emulsion and micellarformulations which form stable complexes with biologically active andthereby facilitate the delivery of the biologically active substances tocells.

[0032] The emulsion formulations of this invention are oil-in-wateremulsions which comprise an aqueous carrier and the following lipidcomponents, an oil component, a cationic amphiphile component, anonionic surfactant component and optionally, a neutral phospholipidcomponent.

[0033] Preferably, the total lipid components are present in theemulsion formulation in an amount from about 0.001 to about 20% byweight, more preferably from about 0.01 to about 10% by weight and mostpreferably from about 0.05 to about 2% by weight, with the remainder ofthe emulsion by weight being aqueous carrier. Thus, for example, forformulation #1 in Table 1 where 0.625 mg of total lipid components arepresent in 0.5 ml of PBS, the weight % of total lipid components informulation #1 can be calculated as follows: Assuming 1 ml of PBS, like1 ml of water, weighs approximately 1000 mg, then 0.5 ml of PBS weighs500 mg and the weight % of total lipid components contained informulation #1 is${\frac{0.625\quad {mg}}{{0.625\quad {mg}} + {500\quad {mg}}} \times 100} = {0.125\quad {\%.}}$

[0034] Of the total lipid components present in the emulsionformulations of this invention, preferably, the amphiphile component ispresent in an amount from about 5 to about 80 weight % of the totallipid components in the emulsion formulation; the oil component ispresent in an amount from about 10 to about 80 weight % of the totallipid components; the nonionic surfactant component is present in anamount from about 5 to about 50 weight % of the total lipid components,and optionally, the neutral phospholipid component is present in theformulation in an amount from about 5 to about 25 weight % of the totallipid component.

[0035] More preferably, the oil component is present in an amount fromabout 10-60 weight % of the total lipid components in the emulsionformulation; the amphiphile component is present in an amount from about20-60 weight % of the total lipid components; the nonionic surfactantcomponent is present in an amount from about 10-50 weight % of the totallipid components and optionally, a neutral phospholipid component ispresent in amount from about 10-40 weight % of the total lipidcomponents.

[0036] Most preferably, the emulsion formulation comprises the oilcomponent in amount from about 10-20 weight % of the total lipidcomponents; the amphiphile component in an amount from about 40-60weight % of the total lipid components; the nonionic surfactantcomponent in an amount from about 20-50 weight % of the total lipidcomponents and optionally, the neutral phospholipid component in anamount from about 10-20 weight % of the total lipid components. Aparticularly preferred emulsion formulation contains an oil, a cationicamphiphile, a nonionic surfactant and a neutral phospholipid in a weightratio of about 2:6:1:2.

[0037] The micellar formulations of this invention are compatible withblood. The micellar formulations comprise an aqueous carrier and thefollowing lipid components: a cationic amphiphile component, a nonionicsurfactant component and optionally, a neutral phospholipid component.

[0038] Preferably, the total lipid components are present in themicellar formulation in an amount ranging from about 0.0001 to about 70%by weight, more preferably from about 0.001 to about. 60% by weight andmost preferably from about 0.001 to about 50 by weight, with theremainder by weight of the micellar formulation being aqueous carrier.Thus, for example, for formulation #15 in Table 2 where 1.25 mg of totallipid components are present in 1 ml of PBS, the weight % of total lipidcomponents in formulation #15 can be calculated as follows: Assuming 1ml of PBS, like 1 ml of water, weighs approximately 1000 mg, then theweight % of total lipid components in formulation #15 is${\frac{1.25\quad {mg}}{{1.25\quad {mg}} + {1000\quad {mg}}} \times 100} = {0.125\quad {\%.}}$

[0039] Of the total lipid components contained in the micellarformulations of this invention, preferably, the amphiphile component ispresent in an amount from about 10 to about 90 weight 0% of the totallipid components in the micellar formulation, the nonionicsurfactant-component is present in an amount from about 10 to about 90weight % of the total lipid components; and optionally, the neutralphospholipid component is present in an amount from about 5 to about 40weight % of the total lipid components.

[0040] More preferably, the amphiphile component is present in an amountfrom about 30 to about 90 weight % of the total lipid components in themicellar formulation, the nonionic surfactant component is present in anamount from about 10 to about 70 weight % of the total lipid componentsand optionally, the neutral phospholipid component is present in anamount from about 5 to about 30 weight % of the total lipid components.

[0041] Most preferably, the amphiphile component is present in an amountfrom about 50 to about 90 weight %C of the total lipid components in themicellar formulation, the nonionic surfactant component is present in anamount from about 10 to about 50 weight % of the total lipid componentsand optionally, the neutral phospholipid component is present in anamount from about 10 to about 20 weight % of the total lipid components.A particularly preferred micellar formulation contains a cationicamphiphile, a nonionic surfactant and a neutral phospholipid in a weightratio of about 6:1:2.

[0042] By oil component as used herein is meant any water immisciblecomponent that is conventionally referred to as an oil. It is understoodthat the oil component may include mixtures of two or more oils.Examples of oils which can be used to produce the emulsion formulationsof the present invention include, but are not limited to, natural oilssuch as almond oil, coconut oil, cod liver oil, corn oil, cottonseedoil, castor oil, olive oil, palm oil, peanut oil, peppermint oil, roseoil, safflower oil, sesame oil, soybean oil, sunflower oil and vegetableoil and synthetic oils such as triethylglycerol and diethylglycerol. Apreferred oil is castor oil.

[0043] The cationic amphiphile component of the formulations of thisinvention may be any cationic amphiphile or mixture of amphiphiles whichis effective for use in liposomes or for producing lipid complexescapable of delivering a biologically active substance to cells. Forexample, the amphiphiles described in Bolcsak et al U.S. Pat. No.5,100,662, which is incorporated herein by reference, would be suitablefor use in this invention. Additional examples of cationic amphiphilessuitable for formulating the emulsion and micellar formulations of thisinvention include, but are not limited to, cationic lipids such as1,2bis(oleoyloxy)-3-(trimethylammonio) propane (DOTAP);N-[1,-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium chloride(DOTMA) or other N-(N,N-1-dialkoxy)-alklyl-N,N,N-trisubstituted ammoniumsurfactants; 1,2dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol(DORT) or cholesteryl (4′trimethylammonia) butanoate (ChOTB) where thetrimethylammonium group is connected via a butanoyl spacer arm to eitherthe double chain (for DOTB) or cholesteryl group (for ChOTB); DORI(DL-1,2-dioleoyl-3-dimethylaminopropyl-B-hydroxyethylammonium) or DORIE(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE)or analogs thereof as disclosed in WO 93/03709, incorporated herein byreference; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC);cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such asdoctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidyesthanolamidospermine (DPPES) or the cationic lipidsdisclosed in U.S. Pat. No. 5,283,185, incorporated herein by reference,cholesteryl-3β-carboxyl-amido-ethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3β-carboxyamidoethyleneamine,cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinateiodide, 2-[(2-trimethylammonio)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinate iodide,3β[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), and3β-[N-(polyethyleneimine)-carbamoyl]cholesterol.

[0044] Examples of preferred amphiphiles includecholesteryl-3β-carboxyamidoethylenetri-methylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3β-carboxyamidoethyleneamine,cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinateiodide,2-[(2-trimethylammonio)ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinateiodide,3β[N-(N′, N′dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol), and3β[N-(polyethyleneimine)-carbamoyl]cholesterol.

[0045] Since an attribute of the emulsion and micellar formulations ofthe present invention is their stability when stored alone or ascomplexes with biologically active substances, it will be understood bythose of ordinary skill in the art that preferred cationic amphiphilesare cationic lipids in which bonds between the lipophilic group and theamino group are stable in aqueous solution. Such bonds include amidebonds, ester bonds, ether bonds and carbamoyl bonds. A more preferredcationic lipid is DC-Chol.

[0046] The nonionic surfactant component of the formulations of thisinvention includes at least one nonionic surfactant of a molecularweight between 200 and 20,000. In one embodiment, these surfactants maybe formed by reacting a hydrophobic hydroxyl-containing compound (e.g.,an alcohol or phenol) with ethylene oxide where the number of ethyleneoxide groups may be added to any desired extent. However, those ofordinary skill in the art would understand that the ability to stabilizethe emulsions or micelles of this invention may depend on the relativeamount of ethylene oxide added to a given hydrophobic group. It isfurther understood that surfactants having branched chain ethylene oxidemoieties cover more surface area than surfactants having single chainethylene oxide moieties and that therefore, the single chain surfactantsmay have to be used in larger amounts than the branched chainsurfactants to produce the emulsion and micellar formulation of thisinvention.

[0047] Examples of nonionic surfactants of this invention include, butare not limited to, polyethylene glycol, derivatives ofphosphatidylethanolamine and synthetic detergents commercially availableunder the brand names Span,

[0048] Preferred surfactants are branched chain surfactants such asTween 20, Tween 40, Tween 60 and Tween 80.

[0049] When optionally added to the emulsion and micellar formulationsof this invention, the neutral phospholipid component way be a singleneutral phospholipid or a mixture of neutral phospholipids. Examples ofneutral phospholipids which may be optionally added to the formulationsof this invention include, but are not limited to, phosphatidylcholine(PC) or phosphatidylethanolamine (PE) or fully saturated or partiallyhydrogenated phosphatidylcholines (PC) or phosphatidylethanolomines (PE)having aliphatic chains between 6 and 24 atoms in length such asdioleoyl-PC (DOPC) and dioleoyl-PE (DOPE). A preferred neutralphospholipid is DOPE.

[0050] Methods for producing the emulsion and micellar formulations ofthe present invention are also provided.

[0051] One method for producing emulsion formulations of this inventioncomprises:

[0052] (a) combining an organic solvent with an oil component, acationic amphiphile component, a nonionic surfactant component andoptionally, a neutral phospholipid component;

[0053] (b) removing the organic solvent to leave a lipid film; and

[0054] (c) suspending the lipid film in an aqueous carrier to producesaid emulsion formulation.

[0055] An alternative method for producing the emulsion formulations ofthis invention comprises:

[0056] (a) combining an oil component, a cationic amphiphile component,a nonionic surfactant component and optionally, a neutral phospholipidcomponent; and

[0057] (b) adding an aqueous carrier to the combination of components instep (a) to produce said emulsion.

[0058] Preferably, average diameters of the emulsion formulations areless than about 1000 nm, more preferably less than 800 nm, and mostpreferably less than 500 nm.

[0059] Preferred components of the emulsions of the present inventioninclude phosphate-buffered saline (PBS) as the aqueous carrier, castoroil as the oil component, DC-Chol as the amphiphile component, Tween 80as the nonionic surfactant component and optionally, phosphatidylcholineor DOPE as the neutral phospholipid component.

[0060] A method for producing the micellar formulations of thisinvention comprises:

[0061] (a) combining an organic solvent with a cationic amphiphilecomponent, a nonionic surfactant component and optionally a neutralphospholipid component;

[0062] (b) removing the organic solvent to leave a lipid film; and

[0063] (c) suspending the lipid film in an aqueous carrier to form saidmicellar formulation.

[0064] Preferably, average diameters of the micellar formulations areless than about 1000 nm, more preferably less than about 800 rim; andmost preferably less than about 500 nm.

[0065] Preferred components of the micellar formulations of the presentinvention include phosphate-buffered saline as the aqueous carrier,DC-Chol as the amphiphile component, Tween 80 as the nonionic surfactantcomponent and optionally, PC or DOPE as the neutral phospholipidcomponent.

[0066] When an organic solvent is used in the above methods to producethe micellar and emulsion formulations of this invention, any organicsolvent which does not leave a toxic residue following removal and whichsolubilizes the lipid components of the emulsion and micellarformulations of this invention is suitable for use. Examples of suitablesolvents include lower alcohols, dimethoxyethane, dioxane,tetrahydrofuran, tetrahydropyran, diethylether, acetone,dimethylsulfoxide (DMSO), dimethylformamides (DMF), and halogenatedhydrocarbons, such as chloroform, acetonitrile, or mixtures thereof. Apreferred organic solvent is chloroform.

[0067] The organic solvent may be removed by drying the combination ofstep (a) under a suitable gas such as argon or nitrogen and/or under avacuum. The dried film may then be lyophilized and stored at about −80to about 37° C. or may be resuspended in a suitable aqueous carrier.Aqueous carriers suitable for use in this invention are non-toxic tocells and may or may not be buffered. When the carriers are buffered,suitable buffers include buffers such as citrate, carbonate,bicarbonate, acetate, Tris, glycinate and maleate. Aqueous carrierswhich may be used in the formulations of this invention include, but arenot limited to, distilled water, normal saline solution andphosphate-buffered saline. It is to be understood that a preferred pHrange for the emulsion and micellar formulations of this invention is apH range in which the particular cationic amphiphile component presentin a formulation is positively charged. Those of ordinary skill in theart would readily be able to determine such a pH range from the pKa ofthe cationic amphiphile component present in a particular formulation.

[0068] It is further understood that the aqueous carrier in which thelipid film is suspended may include ingredients such as stabilizers,antibiotics, or antifungal and antimycotic agents.

[0069] Once formed, the micellar and emulsion formulations may be mixedwith biologically active substances to produce complexes which arestable in storage as reflected by a retention of the activity of thebiologically activity substance over time or by retention of thediameter of the emulsion or micellar formulation over time.

[0070] In one embodiment, the ability of an emulsion or micellarformulation of this invention to deliver a biologically active substanceto a cell may be tested by exposing cells to complexes formed between anemulsion or micellar formulation and a plasmid construct containing areporter gene as the biologically active substance. Such reporter genesare known to those of ordinary skill in the art and include, but are notlimited to, the chloramphenicol acetyltransferase gene, the luciferasegene, the β-galactosidase gene and the human growth hormone gene.

[0071] By “biologically active substance” as used throughout thespecification and claims is meant a molecule, compound, or composition,which, when present in an effective amount, reacts with and/or affectsliving cells and organisms. It is to be understood that depending on thenature of the active substance, the active substance may either beactive at the cell surface or produce its activity, such as with DNA orRNA, after being introduced into the cell.

[0072] Examples of biologically active substances include, but are notlimited to, nucleic acids such as DNA, cDNA, RNA (full length mRNA,ribozymes, antisense RNA, decoys), oligodeoxynucleotides(phosphodiesters, phosphothioates, phosphoramidites, and all otherchemical modifications), oligonucleotide (phosphodiesters, etc.) orlinear and closed circular plasmid DNA; carbohydrates; proteins andpeptides, including recombinant proteins such as for example cytokines(is interleukins), trophic and growth or naturation factors (eg NGF,G-CSF, GM-CSF), enzymes, vaccines (eg HBsAg, gp120); vitamins,prostaglandins, drugs such as local anesthetics (e.g. procaine),antimalarial agents (e.g. chloroquine), compounds which need to crossthe blood-brain barrier such as anti-parkinson agents (e.g. leva-DOPA),adrenergic receptor antagonists (e.g. propanolol), anti-neoplasticagents (e.g. doxorubicin), antihistamines, biogenic amines (e.g.dopamine), antidepressants (e.g. desipramine), anticholinergics (e.g.atropine), antiarrhythmics (e.g. quinidine), antiemetics (e.g.chloroprimamine) and analgesics (e.g. codeine, morphine) or smallmolecular weight drugs such as cisplatin which enhance transfectionactivity, or prolong the life time of DNA in and outside the cells.

[0073] When the biologically active substance is an antigenic protein orpeptide, the complexes formed by the emulsion or micellar formulationsof the present invention may be utilized as vaccines. In thisembodiment, the presence of oil in the emulsion formulation may enhancean adjuvant effect of the complex.

[0074] Preferred biologically active substances are negatively chargedsubstances such as nucleic acids, negatively charged proteins andcarbohydrates including polysaccharides, or negatively charged drugs.

[0075] In a more preferred embodiment the biologically active substancesare nucleic acids and in a most preferred embodiment, the nucleic acidsare nucleic acids which encode a gene or a gene fragment or which effecttranscription and/or translation.

[0076] The complexes of the present invention may be utilized to deliverbiologically active substances to cells in vitro or in vivo.

[0077] When the biologically active substance is a nucleic acid, it isbelieved that the cationic amphiphile binds to the negatively chargednucleic acid. Preferably, nucleic acid:emulsion complexes of thisinvention to be used in vitro or in vivo have a weight ratio of nucleicacid:total lipid components in the emulsion of about 1:1 to about 1:50,more preferably a weight ratio of nucleic acid:total lipid components inthe emulsion of about 1:1 to about 1:30 and most preferably, a weightratio of nucleic acid:total lipid components in the emulsion of about1:1 to about 1:20. Thus for example, in Example 11 where a DNA:emulsioncomplex was formed by combining 100 μg of DNA with a volume of emulsionformulation #21 containing 1100 μg total lipid components, the weightratio of DNA:total lipid components of emulsion #21 in the complex was100 μg/1100 μg or 1:11.

[0078] Preferably, nucleic acid:micelle complexes of this invention tobe used in vitro or in vivo have a weight ratio of nucleic acid:totallipid components in the micelle of about 1:1 to about 1:50, morepreferably a weight ratio of nucleic acid:total lipid components in themicelle about 1:1 to about 1:30 and most preferably a weight of nucleicacid:total lipid components of the micelle ratio of about 1:1 to about1:20. Thus for example, in Example 11 where a DNA:micelle complex wasformed by combining 100 μg of DNA with a volume of micellar formulation#31 containing 700 μg total lipid components, the weight ratio ofDNA:total lipid components of micelle #31 in the complex was 100 μg/700μg or 1:7.

[0079] It is to be understood that the combining of emulsion or micellarformulations with a nucleic acid to form the nucleic acid:emulsion ornucleic acid:micellar complexes of this invention may be carried out forat least 5 minutes in the presence or absence of serum at a temperaturefrom about 4° C. to about 37° C. The resultant nucleic acid:emulsion andnucleic acid:micelle complexes may then be immediately used in vitro orin vivo or may be stored prior to use.

[0080] Preferably, average diameters of nucleic acid:emulsion or nucleicacid:micelle complexes to be used in vitro or in vivo are 100-4000 nm,more preferably 100-2000 nm and most preferably 100-1000 nm.

[0081] In one embodiment, the nucleic acid micelle and nucleicacid:emulsion complexes of this invention may be used to transfect cellswith nucleic acid. Cells suitable for transfection in vitro includeeukaryotic cells, including all mammalian cell lines suitable fortransfection by cationic-lipids, cells put into primary culture from ahost, or cells resulting from passage of the primary culture.

[0082] When, for example, 10⁵ cells are transfected in vitro,transfection is carried out by exposing the cells to preferably fromabout 0.1 to about 5 μgs of nucleic acid:emulsion complex, morepreferably to about 0.5 to from about 2 μgs of nucleic acid:emulsioncomplex.

[0083] When 10⁵ cells are to be transfected with nucleic acid:micellecomplex, transfection is carried out by exposing the cells to preferablyfrom about 0.1 to about 20 μgs of nucleic acid:micelle complex; morepreferably to about 1 to about 10 μgs of nucleic acid:micelle complex.

[0084] As used herein, μg of nucleic acid:emulsion complex or μg ofnucleic acid:micelle complex refers to the sum of the μg amount ofnucleic acid and the μg amount of total lipid components in the emulsionor micellar formulation contained in the complex. For example, 5 μg ofnucleic acid:emulsion complex might contain 0.5 μg of nucleic acid and4.5 μg of total lipid components of emulsion formulation.

[0085] Those of ordinary skill in the art would readily understand thatthe total amount of nucleic acid:emulsion or nucleic acid:micellecomplex to be used varies directly with the number of cells to betransfected. One advantage of the emulsion and micellar formulations ofthis invention over prior art cationic lipid vectors is that theemulsions and micelles of the invention, when complexed with nucleicacid, are more effective for transfecting cells cultured inserum-containing medium.

[0086] The present invention therefore relates to the use of the nucleicacid:emulsion and nucleic acid:micelle complexes of this invention todeliver nucleic acids to cells in an animal or human in vivo. Thus, thepresent invention also relates to the use of nucleic acid:emulsion ornucleic acid:micelle complexes as delivery systems in gene therapy.

[0087] Suitable routes of administration of the nucleic acid containingcomplexes of this invention to an animal or human include inoculation orinjection by, for example, intravenous, oral, intraperitoneal,intramuscular, subcutaneous, intra-aural, intraarticular orintra-mammary routes, topical application, for example on the skin,scalp, ears or eyes and by absorption through epithelial ormucocutaneous linings, for example, nasal, oral, vaginal, rectal andgastrointestinal among others, and as an aerosol. Those of ordinaryskill in the art would readily understand that the mode ofadministration may determine the sites in the organism to which thebiologically active substance will be delivered and may effect theamount of complex to be administered.

[0088] Since as shown in Example 11, administration of approximately 4μg of nucleic acid:emulsion or nucleic acid:micelle complex/gram of bodyweight to a 25 gram mouse produced transfection activity in vivo, thoseof ordinary skill in the art using this ratio of 4 μg of complex/gram ofmouse body weight could obtain other ratios of μg of complex/gram ofbody weight which are optimized for transfection activity in otheranimals or humans.

[0089] In an alternative embodiment, the emulsion and micellarformulations themselves may bind with biomacromolecules (i.e. moleculesproduced by the animal or human) in situ after systematic or topicaladministrations and behave as a local depot for endogenous bioactivesubstances.

[0090] All articles or patents referenced herein are incorporated byreference. The following examples illustrate various aspects of theinvention but are in no way intended to limit the scope thereof.

EXAMPLES Material and Methods

[0091] Materials:

[0092] DC-Chol cationic lipid was synthesized according to Gao and Huang(Biochem. Biophys. Res. Commun., 179:280-285, 1991). Tween 80 and castoroil were obtained from Fisher, pluronic co-polymer L63 was obtained fromBASF. Brij, Span, and pluronic F68 and F127 surfactants were purchasedfrom Sigma. Dioleoyl phosphatidylethanolamine (DOPE) and eggphosphatidylcholine (PC) were obtained from Avanti Polar Lipids.LipofectAMINE liposomes (DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N,-dimethyl-1-propanaminium) and DOPE) in a weightratio of 3:1) were obtained from Life Technologies, Inc.

[0093] Preparation of Emulsions and Micelles:

[0094] Tween 80 diluted in chloroform was combined with DC-Chol(micelles) and, where indicated DOPE or phosphatidylcholine; or withcastor oil, DC-Chol and, where indicated, DOPE or phosphatidylcholine(emulsions) at different weight ratios. The organic solvent was thenevaporated under a stream of nitrogen gas and the lipid film was vacuumdesiccated at 4° C. overnight to remove residual organic solvent. One mlof phosphate buffered saline (PBS, pH 7.4) was then added and themixture was allowed to hydrate for 1 h. The lipid suspension was thenmixed with a vortex mixer and subsequently homogenized for 3-4 min usinga tissue tearer at a speed of about 20,000 rpm. Average diameters of theemulsion of micelle formulations and of the DNA:emulsion or DNA:micellecomplexes were measured by laser light scattering using a Coulter N4SDsubmicron particle sizer.

[0095] Preparation of DC-Chol/DOPE Liposomes:

[0096] Unilamellar small liposomes of approximately 100 nm in diameterwere prepared by microfluidization of hydrated mixture of DC-Chol andDOPE (weight ratio of 1:1) and filter sterilized. The final lipidconcentration of the DC-Chol/DOPE liposomes used in the transfectionexperiments was 1.2 μg/μl of PBS.

[0097] Tissue Culture:

[0098] Murine melanoma BL6 cells were cultured in RPMI mediumsupplemented with 10% fetal bovine serum. Human embryonic kidney 293cells and F_(o) were cultured in DMEM medium supplemented with 10% fetalbovine serum. CHO cells were cultured in F12 medium supplemented with10% fetal bovine serum.

[0099] Plasmid DNA:

[0100] A pCDNA₃ plasmid, pCMV-Luc, containing the luciferase gene underthe control of cytomegalovirus (CMV) immediate early promoter was usedto assess the efficiency of transfection. A similar plasmid, pRSV-Luc,containing the same luciferase gene under the control of a Rous sarcomavirus promoter was also used to assess transfection efficiency. PlasmidpCMV-CAT is a pUC18 based plasmid containing the E. coli chloramphenicolacetyltransferase (CAT) gene downstream from the CMV promoter. Thepreparation and purification of plasmid DNA was carried out according tostandard procedure (Sambrook, J., Fritsch, E. F., & Maniatis, T.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab Press:Plainview, 1: pp 21-52, 1989.).

[0101] Transfection:

[0102] Cells cultured in 48 well plates (about 70-80% confluent) wereused for transfection and 3 wells were transfected with eachformulation. The pCMV-Luc or pRSV-Luc plasmid DNAs were diluted in 125μl of serum free CHO-S-SFM medium (Life Technologies, Inc.). Theemulsion, micelle, DC-Chol/DOPE liposomes or lipofectAMINE liposomeswere diluted in 125 μl of Hank's balanced salt solution (HBSS). Thediluted DNA and formulations or liposomes were combined, with or withoutthe addition of fetal bovine serum to 20%, and incubated at roomtemperature for 5-10 min, before being added to the cells. The cellswere incubated at 37° C. for 5 h. Transfection medium was replaced withgrowth medium containing 10% fetal bovine serum, and cells were thencultured for 2 days before the luciferase assay was performed.

[0103] Luciferase Assay:

[0104] Cells were washed twice with PBS and incubated at roomtemperature for 10 min in the presence of 100 μl lysis buffer (0.1MTris-HC-1, pH 7.8/0.05% Triton X-100/2 mM EDTA) and then centrifuged at12,000×g. Ten μl of supernatant was taken for luciferase assay using theluciferase assay system (Promega) in a luminometer (AutoLumat LB953 fromEG&G, Berthhold). Luciferase activity is given in relative light units(RLU).

[0105] Animal Studies:

[0106] Female CD 1 mice, 5 weeks old, were purchased from Charles RiverBreeding Laboratories. Animal care was according to the institutionalguidelines. 200 μl each of 4× concentrates (for example, the 4×concentrate of formulation #21 contained 1.0 mg oil, 1.0 mg PC, 0.5 mgTween 80 and 3.0 mg DC-Chol per ml of PBS) of formulations #21 (1100 μgtotal lipid components), #28 (1100 μg total lipid components), #34 (900μg total lipid components) and #31 (700 μg total lipid components) (600μg DC-Chol per formulation) were mixed with 5M NaCl to a finalconcentration of NaCl of 0.15M and then combined with 25 μl of 4 μg/μlpCMV-CAT DNA (100 μg). The total mixture (231 μl) was injected into miceby tail vein. Two days later, mice were killed and liver, spleen,kidney, lung and heart were excised for CAT assay.

[0107] Chloramphenicol Acetyltransferase(CAT) Assay:

[0108] The organs excised from animals were homogenized in 40 mMTris-HCl, pH 7.5;10 mM EDTA; 150 mM NaCl. After homogenization, cellswere lysed by three freeze-thaw cycles, and the lysate was heated at 65°C. for 10 min to inactive deacetylases and centrifuged for 10 min. Theprotein concentration of the supernatant extracts was measured with aCoomassie blue G 250-assay (Pierce). Protein was extracted from eachorgan and 200 μg of extract was then assayed for the CAT activity using[¹⁴C] chloramphenicol as a substrate as previously described (Ausubel,F. M., Breht, R., Kingstone, R. E., et al. Current Protocols inMolecular Biology (Wiley, Boston), Vol. 1, pp 962-965, 1991.).

Example 1 Physical Stability of Emulsion Formulations and TransfectionAbility of DNA:Emulsion Complexes

[0109] To test which components of the emulsion formulations areimportant for physical stability and transfection ability, 9 differentemulsion formulations containing different amounts of castor oil, eggphosphatidylcholine (PC), Tween 80 and DC-Chol were formulated. Theaverage diameter of the formulations was measured as was their abilityto transfect 293 cells by combining 6 μl of each emulsion (7.5 μg totallipid components) with 0.5 μg RSV-Luc. The resulting data are presentedin Table 1. TABLE 1 Transfection activity of DNA:emulsion complexesformed by combining DNA with different emulsion formulations Composition(mg) Average of formulation Diameter (nm) Transfection Activity^(b)Formulation Oil PC Tween 80 DC-Chol PBS (ml)^(a) of formulation (RLU ×10⁶/well) #1 0.125 0.250 0.125 0.125 0.50 170 118 ± 22  #2 0.250 0.2500.250 0.750 1.20 151 444 ± 4  #3 0.750 0.250 0.025 0.250 1.02 212 265 ±46  #4 0.125 0.125 0.025 0.750 0.82 218 332 ± 89  #5 0.250 0.125 0.1250.250 0.60 181 220 ± 7  #6 0.750 0.125 0.250 0.125 1.00 154 68 ± 1  #70.125 0.500 0.250 0.250 0.90 165 65 ± 20 #8 0.250 0.500 0.025 0.125 0.72201   1 ± 0.6 #9 0.750 0.500 0.125 0.750 1.70 161 261 ± 47  DC-Chol/DOPE122 445 ± 2  (1:1 ratio by weight) LipofectAMINE (DOSPA and 100 427 ±25  DOPE in a 3:1 weight ratio) #2.25 μl of LipofectAMINE liposomes (4.5μg lipid components). Each well contained approximately 70‥80 μgextractable protein.

[0110] The data show that the emulsions are physically stable with sizeranging from 150 to 218 nm in average diameter as measured by laserlight scattering. Further, complexes of DNA with those emulsions withhigh content of DC-Chol (0.750 mg), the only cationic component in theemulsion, showed high transfection activity comparable to that of thecationic DC-Chol/DOPE and LipofectAMINE liposomes.

Example 2 Transfection Activity of DNA:Emulsion and DNA:MicelleComplexes

[0111] Emulsion and micellar formulations which contained high contentof the cationic amphiphile DC-Chol (0.75 mg or more) were complexed withpRSV-Luc DNA and examined for transfection activity in BL6 and 293cells. The data presented in Table 2 TABLE 2 Transfection activity ofDNA:emulsion and DNA:micelle complexes Average Composition (mg) DiameterTransfection Activity^(b) of formulation (nm) of BL6 cells 293 cellsFormulation Oil PC Tween 80 DC-Chol DOPE Stearylamine PBS (ml)^(a)formulation (RLU × 10³/well) (RLU × 10⁶/well) #10 0.250 0.250 0.2500.750 — — 1.2 129 13 ± 8 530 ± 78 #11 0.125 0.125 0.250 0.375 — — 0.7143 15 ± 4 546 ± 62 #12 0.125 0.125 0.375 0.375 — — 0.8 127  4 ± 2 239 ±15 #13 0.125 0.125 0.125 0.500 — — 0.7 153  54 ± 34  708 ± 213 #14 0.1250.125 0.125 0.750 — — 0.9 152 112 ± 37 880 ± 13 #15 — 0.250 0.250 0.750— — 1.0 165 19 ± 2 861 ± 22 #16 0.250 — 0.250 0.750 — — 1.0 161  78 ± 14710 ± 47 #17 0.250 — 0.250 0.750 0.250 — 1.2 155  23 ± 10 463 ± 90 #180.250 0.250 0.250 — — 0.400 0.90 193  3 ± 1  9 ± 1 #19 — — 0.250 0.750 —— 0.8 186  262 ± 104 806 ± 71 #20 0.250 0.250 — 0.750 — — 1.0 160 19 ± 9840 ± 20 DC-Chol/DOPE 0.600 0.600 1.0 122  81 ± 40 221 ± 35#approximately 70-80 μg extractable protein except for #18 whichcontained 50 μg protein.

[0112] show that complexes of DNA with the emulsions and the twomicellar formulations (#15 and #19) were active. However, the complexformed by combining DNA with formulation #18 that contained stearylamineinstead of DC-Chol showed low transfection activity which may be due tothe toxicity of stearylamine to cells as evidenced by the fact that theamount of protein extractable from wells transfected with complexcontaining formulation #18 was less than the amount of proteinextractable from wells transfected with complexes containing all otherformulations (see Table 2, footnote b). Of interest, the activities of amicellar (#19) and an emulsion (#14) DNA complex were high, and indeedmore active, than the cationic liposome formulation (Dc-Chol/DOPE) inboth cell lines.

Example 3 Transfection Activity of More DNA:Emulsion and DNA:MicelleComplexes

[0113] The physical diameter of additional emulsion and micellarformulations was measured as was the transfection activity of complexesformed between DNA and these formulations. The results are shown inTable 3. TABLE 3 Transfection activity of more DNA:emulsion andDNA:micelle complexes Composition (mg)^(a) of formulation AverageTransfection Activity^(b) Pluronic Diameter (nm) BL6 293 Formulation OilPC Tween 80 DC-Chol DOPE L63 of formulation (RLU × 10⁵/well) (RLU ×10⁷/well) #21 0.250 0.250 0.125 0.750 — — 137 228 ± 40 — #22 0.250 0.2500.250 0.750 — — 218 235 ± 67 101 ± 10 #23 0.250 0.250 0.500 0.750 — —133   2 ± 0.4  11 ± 16 #24 0.250 0.250 0.250 1.000 — — 152 322 ± 76 104± 4  #25 0.250 0.250 0.250 1.500 — — 152  98 ± 28  34 ± 14 #26 — 0.2500.250 0.750 — — 168 450 ± 11 122 ± 40 #27 0.250 — 0.125 0.750 — — 163426 ± 83 130 ± 26 #28 0.250 — 0.125 0.750 0.250 — 169  645 ± 219 34 ± 1#29 0.250 0.250 — 0.750 — — 146 117 ± 44 56 ± 4 #30 — — 0.250 0.750 — —202 759 ± 65  94 ± 58 #31 — — 0.125 0.750 — — 179 324 ± 22 188 ± 40 #32— — — 0.750 — 0.250 204 150 ± 45  88 ± 20 #33 — — — 0.750 — 0.500 212107 ± 32  82 ± 16 #34 — — 0.125 0.750 0.250 — 200 890 ± 35 —DC-Chol/DOPE 122 1033 ± 204  40 ± 13

[0114] The results show that complexes of DNA and micelles containingDC-Chol and Tween 80 (#30 and #31), were again quite active andreplacing Tween 80 with pluronic L63 (#32 and #33) did not significantlyalter the average diameters of the formulations or their transfectionactivity in 293 cells. Another micelle containing DC-Chol, Tween 80 andPC (phosphatidylcholine) (#26) was also active. The remainingformulations (#22-25, 27-29) were emulsions which contained castor oil.Complexes of DNA and these emulsion formulations were fairly active intransfection. When complexes of DNA and these micellar and emulsionformulations were tested in another cell-line (BL6 mouse melanomacells), qualitatively similar results were obtained except the activityin this cell line was generally lower than that of the 293 cells. Thetransfection activities of complexes of DNA and these emulsions andmicelles were comparable to that of the cationic liposome formulation(DC-Chol/DOPE) in 293 cells, but somewhat lower in BL6 cells.

Example 4

[0115] Optimization of Transfection Conditions:

[0116] To optimize the transfection activity of the DNA:emulsion andDNA:micelle complexes, the amount of DC-Chol in each emulsion or micelleformulation was kept constant at 4.5 μg (6 μl of formulations #s 21, 27,28 and 30, respectively were used; refer to Table 3 for compositions offormulations) and the amount of pCMV-Luc DNA was varied from 0 to 5 μg.As shown in FIG. 1, complexes of DNA and formulations #27 and #30 (referto Table 3 for compositions) showed a maximal transfection activity inBL6 cells at 2 μg DNA while complexes of DNA and formulations #21 and#28 showed a maximum activity at 1.5 μg DNA.

[0117] Next, the amount of pCMV-Luc DNA was fixed at 2 μg forformulations #27 and #30 and at 1.5 μg for formulations #21 and #28 andcomplexes of DNA and varying amounts of emulsion or micelle formulationas indicated on the horizontal axis of FIG. 2 (where μg formulation onthe horizontal axis refers to μg total lipid components present in thevolume of formulation combined with pCMV-Luc DNA to form complex) wereproduced. The results presented in FIG. 2 show that complexes of DNA andformulations #27, #28 and #30 exhibited a relatively broad peak ofactivity in BL6 cells with the optimal amount of total lipid componentspresent in the volume of formulation combined with DNA to producecomplex being about 18 μg. However, complexes of DNA and formulation #21exhibited a narrower peak of activity with the optimal amount of totallipid components present in the volume of formulation combined with DNAto produce complex being about 13 μg.

Example 5 Sensitivity of Transfection Activity of DNA:Emulsion andDNA:Micelle Complexes to Serum

[0118] All the above described transfection experiments were carried outin a serum-free medium. Therefore, to determine the sensitivity oftransfection activity of DNA/emulsion and DNA/micelle complexes to thepresence of serum, BL6 cells were transfected in the medium containing 0or 20% fetal bovine serum with complexes of DNA and 10 differentemulsions or micelles (or DC-Chol/DOPE liposomes) as follows.

[0119] Different formulations of the compositions shown in Table 4 wereprepared in a total volume of 1 ml PBS (pH 7.4). BL6 cells in a 48 wellplate were then transfected with 2 μg of pCMV-Luc and 16 μl of eachformulation (containing 12 μg DC-Chol), or with 2 μg of pCMV-Luc and 16μl of DC-Chol/DOPE liposomes (1.2 μg/μl), in medium containing either 0or 20% fetal bovine serum and luciferase activity was detected. Eachwell contained approximately 70-80 μg extractable protein. The resultsare shown in Table 4 TABLE 4 Transfection Activity Of AdditionalDNA:emulsion and DNA:micelle complexes COMPOSITION (mg)^(a) LUCIFERASEACTIVITY of formulation (RLU/well × 10⁷) FORMULATION Oil PC DOPE Tween80 DC-Chol −Serum +Serum (20%) #35 0.25 0.25 — 0.125 0.75 14 ± 3  49 ±2  #36 — 0.25 — 0.125 0.75 32 ± 6  98 ± 6  #37 0.25 — — 0.125 0.75 170 ±7  5 ± 3 #38 0.25 0.25 — — 0.75  24 ± 0.8 56 ± 13 #39 0.25 0.25 — 0.125— 0 0 #40 0.25 — 0.25 0.125 0.75 50 ± 3  151 ± 6  #41 0.25 — 0.25 — 0.7540 ± 5  85 ± 8  #42 — — 0.25 0.125 0.75 140 ± 5  267 ± 44  #43 — — —0.125 0.75 156 ± 13  298 ± 42  #44 — — — 0.25 0.75 259 ± 12  307 ± 8 DC-Chol/DOPE liposomes — — 0.60 — 0.60 107 ± 21  47 ± 9 

[0120] demonstrate that the transfection activity of DC-Chol/DOPEliposomes was quite sensitive to serum (only about 33% activity remainedin the presence of serum) while of the 10 formulations tested, onlycomplex of DNA and formulation #37 showed serum sensitivity where serumsensitivity is a reduction in transfection activity in the presence ofserum relative to the level of activity observed in the absence ofserum. In addition, the fact that complex of DNA and formulation #39showed no activity in the presence or absence of serum demonstrated thatthe presence of cationic amphiphile is critical to transfectionactivity. Particularly interesting are complexes of DNA and formulations#35, #36 and #40 (corresponding in composition to formulations 21, #34and #28 respectively in Table 3) which, of the formulations tested,exhibited the greatest enhancement of transfection activity in thepresence of serum.

Example 6 Sensitivity of Transfection Activity of Complexes of DNA andSelected Formulations to Serum in Different Cell Lines

[0121] To determine if the serum sensitivity observed in BL6 cells inTable 4 was observed in other cell lines, F_(o) cells, CHO cells and 293cells were transfected with 16 μl of selected formulations (eachcontaining 12 μg DC-Chol/16 Al) from Table 4 combined with 2 μg ofpCMV-Luc DNA or, with 16 μl DC-Chol/DOPE liposomes (1.2 μg/μl) combinedwith 2 μg of pCMV-Luc DNA, in medium containing 0 or 20% serum as inExample 5. The results of these experiments are shown in Table 5. Thedata show that complexes of DNA and all formulations are active intransfecting cells and are in general, serum-resistant. TABLE 5Transfection Activity of Complexes Of DNA And Selected Formulations inDifferent Cell Lines. LUCIFERASE ACTIVITY (RLU/Well × 10⁷) FORMULATIONF₀ Cells CHO Cells 293 Cells (w/w) −Serum +Serum −Serum +Serum −Serum+Serum #35 (Oil/PC/Tw/DC-Chol 8.3 ± 3.7 13.2 ± 7.0 8.0 ± 0.7 5.9 ± 0.433.0 ± 7.8  37.0 ± 3.4 (2:2:1:6)^(a) #37 (Oil/Tw/DC-Chol) 2.7 ± 0.2  1.6± 0.9 1.6 ± 1.6 10.4 ± 1.3  33.3 ± 11.3 44.2 ± 1.2 (2:1:6:)^(b) #40(Oil/DOPE/Tw/DC-Chol) 8.7 ± 2.1 12.0 ± 2.1 0.4 ± 0.0 7.5 ± 0.4 27.9 ±7.0  34.7 ± 2.1 (2:2:1:6)^(c) #44 (Tw/DC-Chol) 22.0 ± 3.3  13.2 ± 1.10.4 ± 0.1 1.5 ± 0.3 68.0 ± 17.0 168.0 ± 17.0 (2:6)^(d) DOPE/DC-Cholliposomes 2.7 ± 0.2  0.5 ± 0.1 1.7 ± 0.0 2.8 ± 0.5 88.0 ± 3.9  67.0 ±3.9 (1:1)^(e)

Example 7

[0122] Stability of DNA:Emulsion and DNA:Micelle Complexes

[0123] Five different formulations (#'s 26, 27, 28, 29 and 34 of Table3) were tested for the stability of their complex with DNA. Complex wasprepared by combining 2 μg pCMV-CAT DNA and 16 μl of the indicatedemulsion or micelle formulation (where 16 μl of each formulationcontained the same amount of DC-Chol, 12 μg) or by combining 1 μgpCMV-CAT DNA and 6 μg of DC-Chol/DOPE liposomes. As can be seen in FIG.3, formulations #26 #28 and #29 formed relatively small complexes withDNA; the average diameter of the complex as measured by laser lightscattering ranged from 200-300 nm, and remained small even after 10 daysat 4° C. Formulation #34 and #27, on the other hand, formed largercomplexes with DNA with average diameters of 600 and 900 nm,respectively. In contrast, DC-Chol/DOPE liposomes had formed largeaggregates (1,800 nm on day 1) which had grown to even larger ones(>4,000 nm) on day 3 and subsequently precipitated out of solution (datanot shown). Thus, all new formulations could form complexes with DNAthat had physical Stability better than that of complexes formed withthe DC-Chol/DOPE liposomes.

Example 8 Effect of Different Surfactants on the Transfection Activityof Complexes of DNA and Emulsions Composed ofOil/DOPE/DC-Chol/Surfactant in a Weight Ratio of 2:2:6:x

[0124] Emulsions containing different surfactants were prepared in 1 mlof PBS containing 0.25 mg of oil, 0.25 mg of DOPE, 0.75 mg of DC-Choland different amounts of the indicated surfactants where the totalamount of surfactant used in each formulation was approximately the sameby mole. BL6 cells were then transfected with 2 μg of pCMV-Luc DNAcombined with 16 μl of formulation (12 μg DC-Chol/16 μl of eachformulation) and assayed for luciferase activity. The results of thisexperiment are shown in Table 6. TABLE 6 Effect Of Different SurfactantsOn The Transfection Activity Of Complexes Of DNA and Emulsions ComposedOf OIL/DOPE/DC- Chol/surfactant (0.25 mg:0.25 mg:0.75 mg:X mg per ml ofPBS) LUCIFERASE ACTIVITY (RLU/Well) × 10⁷ Surfactant X (mg) −Serum+Serum (20%) Tween  20 0.117 1.9 ± 0.4 4.9 ± 1.1  40 0.122 1.9 ± 0.3 5.3± 0.7  60 0.125 3.2 ± 0.3 5.6 ± 2.0 Brij  72 0.034 7.0 ± 0.7 10.0 ± 2.0  74 0.068 9.4 ± 1.6 6.5 ± 0.3  76 0.110 5.4 ± 0.8 0.08 ± 0.03 100 0.4460.3 ± 0.2 0.003 ± 0.006 Span  20 0.033 1.2 ± 0.0 0.1 ± 0.0  40 0.038 1.9± 0.1 0.1 ± 0.0  60 0.041 1.4 ± 0.4 0.3 ± 0.1  80 0.041 1.4 ± 0.4 0.3 ±0.1 pluronic F 0.802 7.8 ± 0.3 9.0 ± 1.4  68 pluronic F 1.202 7.9 ± 0.99.9 ± 1.1 127

[0125] Of the surfactants tested, complexes formed from emulsionscontaining Tween 20, Tween 40, Tween 60, Brij 72, Brij 74, F68 or F127demonstrated transfection activity that was not sensitive to thepresence of 20% serum and complexes formed from formulations containingthe Tween series of detergents showed the greatest increase intransfection activity in the presence of serum relative to that observedin the absence of serum.

Example 9 Effect of Tween 80 Concentration in Emulsions on the AverageDiameters of Concentrated and Diluted DNA/Emulsion Complexes

[0126] Concentrated DNA/emulsion complex was formed by adding 2 μl ofsolution containing 8 μg of DNA (pCMV-Luc) directly to 72 μl ofemulsions containing 0.25 mg Oil/0.25 mg DOPE/0.75 mg DC-Chol andvarying mg amounts of Tween 80 per ml. As in the prior examples dilutedDNA/emulsion complex was formed by combining 2 μg of DNA in 125 μl with18 μl of the same emulsions used in the concentrated complex but dilutedto 125 μl. The average diameters of the concentrated and dilutedcomplexes were measured 1 hour after incubation at room temperature. Theresults shown in FIG. 4 demonstrate that increasing amounts of Tween 80reduced the size of the concentrated complexes but had no effect on thesize of the diluted complexes.

Example 10 Effect of the Amount of Tween 80 in an Emulsion onTransfection Activity of Concentrated and Diluted DNA/Emulsion Complexesin the Presence or Absence of 20% Serum

[0127] Emulsions and concentrated and diluted pCMV-Luc DNA/emulsioncomplexes were prepared as in Example 9 and the transfection activity ofthe complexes was measured in BL6 cells in the presence or absence of20% serum. The results of these experiments are shown in FIGS. 5A(concentrated complex) and 5B (diluted complex). While the dilutedcomplexes appear to show better activity than the concentratedcomplexes, the need to keep the volume of complex administered to ananimal small may favor the use of more concentrated complexes in vivo.

Example 11 Animal Studies With DNA:Emulsion and DNA:Micelle Complexes

[0128] Formulations #21, 28, 31 and 34 (refer to Table 3 forcompositions) were tested for gene transfer activity in mice. 200 μleach of 4× concentrates of formulations #21 (1100 μg total lipidcomponents), #28 (1000 μg total lipid components), #34 (900 μg totallipid components) and #31 (700 μg total lipid components)) were mixedwith 6 μl of 5M NaCl to a final concentration of 0.15M NaCl and thencombined with 4 μg/μl pCMV-CAT DNA (100 μg). The complexes were theninjected i.v. via the tail vein of the mouse (each mouse weighedapproximately 25 grams) and CAT activity was measured in major organstwo days after injection. The data presented in FIG. 6 clearlydemonstrates that complexes of DNA and formulations #28 (emulsion), #31(micelle) or #34 (micelle) could transfect various organs withrelatively high activity while complex of DNA and formulation #21(emulsion), on the other hand, was weak and comparable to that ofDC-Chol/DOPE liposomes. In addition, the activity of complex of DNA andformulation #31 seems to be lung specific, as no other organs weresignificantly transfected; complex of DNA and formulation #28 couldtransfect all organs quite well with only weak transfection of thekidney, and complex of DNA and formulation #34 showed a high activity inthe heart with very low activity in the kidney.

1. An oil-in-water emulsion formulation comprising lipid components andan aqueous carrier, wherein the lipid components comprise an oilcomponent, a cationic amphiphile component and a nonionic surfactantcomponent.
 2. The emulsion formulation of claim 1 wherein the lipidcomponents further comprise a neutral phospholipid component.
 3. Theemulsion formulation of claim 1 wherein the oil component is present inan amount from about 10 to about 80 weight % of the total lipidcomponents in the formulation, the amphiphile component is present in anamount from about 5 to about 80 weight % of the total lipid componentsand the nonionic surfactant component is present in an amount from about5 to about 50 weight % of the total lipid components.
 4. The emulsionformulation of claim 3 further comprising a neutral phospholipidcomponent present in an amount from about 5 to about 25 weight % of thetotal lipid components in the formulation.
 5. The emulsion formulationof claim 4, wherein the amphiphile component is a cationic lipid.
 6. Amicellar formulation comprising lipid components and an aqueous carrier,wherein the lipid components comprise a cationic amphiphile componentand a nonionic surfactant component.
 7. The micellar formulation ofclaim 6, wherein the lipid components further comprise a neutralphospholipid component.
 8. The micellar formulation of claim 6 whereinthe amphiphile component is present in an amount from about 10 to about90 weight % of the total lipid components and the nonionic surfactantcomponent is present in an amount from about 90 to about 10 weight % ofthe total lipid components.
 9. The micellar formulation of claim 8,further comprising a neutral phospholipid component present in an amountfrom about 5 to about 40 weight % of the total lipid components.
 10. Themicellar formulation of claim 9, wherein the amphiphile component is acationic lipid.
 11. A complex for facilitating the delivery of anegatively charged biologically active substance to cells, said complexcomprising the negatively charged biologically active substance and theemulsion formulation of claim
 1. 12. The complex of claim 11, whereinthe negatively charged substance is a nucleic acid.
 13. The complex ofclaim 12, wherein the weight ratio of nucleic acid to total lipidcomponents in the emulsion formulation in said complex is about 1:1 toabout 1:50.
 14. A complex for facilitating the delivery of a negativelycharged biologically active substance to cells, said complex comprisingsaid negatively charged substance and the micellar formulation of claim6.
 15. The complex of claim 14, wherein the negatively charged substanceis a nucleic acid.
 16. The complex of claim 15, wherein the weight ratioof nucleic acid to total lipid components in the micellar formulation insaid complex is from about 1:1 to about 1:50.
 17. A method fordelivering a negatively charged biologically active substance to cellscomprising exposing the cells to the complex of claim 11 therebyfacilitating the delivery of the negatively charged biologically activesubstance to the cells.
 18. The method of claim 17 wherein the cells aremammalian cells exposed to the complex in the presence of serum.
 19. Amethod for delivering a negatively charged biologically active substanceto cells comprising exposing the cells to the complex of claim 14thereby facilitating the delivery of the negatively charged biologicallyactive substance to the cells.
 20. The method of claim 19 wherein thecells are mammalian cells exposed to the complex in the presence ofserum.
 21. The methods for delivering a negatively charged biologicallyactive substance to cells according to claims 17 and 19 wherein thecells are exposed to the complex in vivo by administering the complexesto an animal or human in an amount effective to facilitate the deliveryof the negatively charged substance to the cells of the animal or human.22. The method of claim 21, wherein the negatively charged substance isa nucleic acid.
 23. The methods for delivering a negatively chargedbiologically active substance to cells according to claims 17 and 19wherein the cells are exposed to the complex in vitro.
 24. The method ofclaim 23, wherein the negatively charged substance is a nucleic acid.25. A method of producing an oil-in-water emulsion formulationcomprising: (a) combining an oil component, a cationic amphiphilecomponent and a nonionic surfactant component; and (b) adding aqueouscarrier to produce said emulsion formulation.
 26. A method of producinga micellar formulation comprising: (a) combining a cationic amphiphilecomponent and a nonionic surfactant component; and (b) adding aqueouscarrier to produce said micellar formulation.
 27. The methods of claims25 and 26 further comprising combining the components of step (a) with aneutral phospholipid component.
 28. The method of claim 27, wherein thecomponents of step (a) are combined in an organic solvent and thesolvent is removed to leave a lipid film prior to step (b).
 29. A methodof producing a lipid film having an oil component, a cationic amphiphilecomponent and a nonionic surfactant component; said method comprising:(a) combining an organic solvent with the oil component, the amphiphilecomponent and the nonionic surfactant component; and (b) removing theorganic solvent to leave said lipid film.
 30. A method of producing alipid film having a cationic amphiphile component and a nonionicsurfactant component; said method comprising: (a) combining an organicsolvent with the amphiphile component and the nonionic surfactantcomponent; and (b) removing the organic solvent to leave said lipidfilm.
 31. A lipid film capable of forming an oil-in-water emulsion uponsuspension in an aqueous carrier, said film having an oil component, acationic amphiphile component and a nonionic surfactant component.
 32. Alipid film capable of forming a micelle upon suspension in solution,said film having a cationic amphiphile component and a nonionicsurfactant component.
 33. The lipid films of claims 32 and 33, saidfilms further having a neutral phospholipid component.